title: Suppression and epigenetic regulation of MiR-9 contributes to ethanol teratology: evidence from zebrafish and murine fetal neural stem cell models.
abstract: BACKGROUND
  Fetal alcohol exposure produces multiorgan defects, making it difficult to identify underlying etiological mechanisms. However, recent evidence for ethanol (EtOH) sensitivity of the miRNA miR-9 suggests one mechanism, whereby EtOH broadly influences development. We hypothesized that loss of miR-9 function recapitulates aspects of EtOH teratology.
  
  
  METHODS
  Zebrafish embryos were exposed to EtOH during gastrulation, or injected with anti-miR-9 or nonsense control morpholinos during the 2-cell stage of development and collected between 24 and 72 hours postfertilization (hpf). We also assessed the expression of developmentally important, and known miR-9 targets, FGFR-1, FOXP2, and the nontargeted transcript, MECP2. Methylation at CpG islands of mammalian miR-9 genes was assessed in fetal murine neural stem cells (mNSCs) by methylation-specific PCR, and miRNA processing assessed by qRT-PCR for pre-miR-9 transcripts.
  
  
  RESULTS
  EtOH treatment and miR-9 knockdown resulted in similar cranial defects including microcephaly. Additionally, EtOH transiently suppressed miR-9, as well as FGFR-1 and FOXP2, and alterations in miR-9 expression were correlated with severity of EtOH-induced teratology. In mNSCs, EtOH increased CpG dinucleotide methylation at the miR-9-2 locus and accumulation of pre-miR-9-3.
  
  
  CONCLUSIONS
  EtOH exerts regulatory control at multiple levels of miR-9 biogenesis. Moreover, early embryonic loss of miR-9 function recapitulated the severe range of teratology associated with developmental EtOH exposure. EtOH also disrupts the relationship between miR-9 and target gene expression, suggesting a nuanced relationship between EtOH and miRNA regulatory networks in the developing embryo. The implications of these data for the expression and function of mature miR-9 warrant further investigation.
authors.affiliation: Department of Neuroscience and Experimental Therapeutics, Texas A&M Health Science Center, College of Medicine, Bryan, Texas.
authors.forename: Dana L
authors.initials: DL
authors.lastname: Pappalardo-Carter
authors.forename: Sridevi
authors.initials: S
authors.lastname: Balaraman
authors.forename: Pratheesh
authors.initials: P
authors.lastname: Sathyan
authors.forename: Eric S
authors.initials: ES
authors.lastname: Carter
authors.forename: Wei-Jung A
authors.initials: WJ
authors.lastname: Chen
authors.forename: Rajesh C
authors.initials: RC
authors.lastname: Miranda
mesh_terms: D000818:Animals; D044127:Epigenesis, Genetic / Q000187:drug effects / Q000502:physiology*; D000431:Ethanol / Q000633:toxicity*; D053686:Fetal Stem Cells / Q000187:drug effects / Q000502:physiology*; D055785:Gene Knockdown Techniques / Q000379:methods; D051379:Mice; D008810:Mice, Inbred C57BL; D035683:MicroRNAs / Q000502:physiology*; D058953:Neural Stem Cells / Q000187:drug effects / Q000502:physiology*; D011897:Random Allocation; D064793:Teratogenesis / Q000187:drug effects / Q000502:physiology*; D015027:Zebrafish
chemical_list: C551804:MIRN9 microRNA, mouse; D035683:MicroRNAs; D000431:Ethanol
keywords: Embryonic Development; Ethanol; Methylation; MiR-9; Zebrafish